Use of a molecular beacon based fluorescent method for assaying uracil DNA glycosylase (Ung) activity and inhibitor screening

Abstract

Uracil DNA glycosylases are an important class of enzymes that hydrolyze the N-glycosidic bond between the uracil base and the deoxyribose sugar to initiate uracil excision repair. Uracil may arise in DNA either because of its direct incorporation (against A in the template) or because of cytosine deamination. Mycobacteria with G, C rich genomes are inherently at high risk of cytosine deamination. Uracil DNA glycosylase activity is thus important for the survival of mycobacteria. A limitation in evaluating the druggability of this enzyme, however, is the absence of a rapid assay to evaluate catalytic activity that can be scaled for medium to high-throughput screening of inhibitors. Here we report a fluorescence-based method to assay uracil DNA glycosylase activity. A hairpin DNA oligomer with a fluorophore at its 5′ end and a quencher at its 3′ ends was designed incorporating five consecutive U:A base pairs immediately after the first base pair (5′ C:G 3’) at the top of the hairpin stem. Enzyme assays performed using this fluorescent substrate were seen to be highly sensitive thus enabling investigation of the real time kinetics of uracil excision. Here we present data that demonstrate the feasibility of using this assay to screen for inhibitors of Mycobacterium tuberculosis uracil DNA glycosylase. We note that this assay is suitable for high-throughput screening of compound libraries for uracil DNA glycosylase inhibitors.

Viruses with U-DNA: New Avenues for Biotechnology

“…A specific example for UNG importance is observed in, e.g., Mycobacterium tuberculosis , where UNG plays a crucial role in maintaining the integrity of its genome especially during in vivo growth because of the exposure of the pathogen to reactive nitrogen intermediates and reactive oxygen intermediates discharges by the host macrophages. These reagents lead to the deamination of cytosine bases of the bacteria GC rich (median GC%: 65.6) genome [ 37 ]. In addition, UNG is among the most important factors limiting the efficiency of antifolates and fludarabine.…”

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

“…Detect caspase-3 and survivin mRNA, [26] Uracil DNA glycosylases (Ung) [35] /MMP-3, [30] /MMPs [39] /MMP-3, [31] hypoxic tissues [41] Detect miR-21, [44] Zika-specific DNA in human blood serum, [45] the activity of anti-digoxigenin (Dig) antibodies, [46] intracellular target mRNA [47] /caspase-3 [48] IQ 4 587 520-706 56 000 -/--/-Eclipse 522 460-570 -/-FAM/aminoacridine Detect Guignardia citricarpa, [50] human immunodeficiency virus type 1 (HIV-1) in plasma [51] /single base pair mutations [52] TAMRA 544 470-560 85 000 FAM Detect AFB1 in beer, wine samples, [58] the mutation level of Kisten rat sarcoma (KRAS) viral gene [59] QSY7 560 500-600 92 000 Cy3/BODIPY/fluorescein (92 300; 518) Detect Cathepsin B (CaB), [67] caspase-3 [75] /E. coli outer membrane protease (OmpT) [72] /protein/ribosome conformation [76] QSY9 562 500-600 88 000…”

“…The same group utilized the Cy5.5/BHQ-3 pair to prepare another MMP-3 fluorogenic probe. [31] In addition to the abovementioned examples, BHQs have also been used in the detection of ATP, [32] esterase, [33] DNA topoisomerase, [34] Uracil DNA glycosylases (Ung), [35] nicking enzyme, [36] conformational changes of ribosome, [37] DNA methylation, [38] MMP-13, [39] cell differentiation of mesenchymal stem cells (MSCs), [40] hypoxic tissues, [41] and Parkinson's disease (PD) biomarkers. [42] [26] with permission.…”